PROJECT SUMMARY/ABSTRACT The goal of this project is to develop GABP as a therapeutic target to reverse immortality of tumors harboring a mutant telomerase reverse transcriptase (TERT) promoter. TERT promoter mutation is the third most common mutation in human cancer, affecting over 80% of GBM and oligodengroglioma (OD). Due to a lack of TERT transcription in somatic cells, telomeres shorten with each successive cell division until they reach a critical level that triggers senescence and limits cell lifespan. Reactivation of TERT expression overcomes these barriers, enabling tumor cells to proliferate indefinitely. Although proteins controlling mutant TERT promoter reactivation and tumor cell immortalization may be ideal therapeutic targets, the exact identity of these molecules remained unknown. We discovered that a single ubiquitously expressed transcription factor, GABP, uniquely bound to the mutant TERT promoter and drove TERT reactivation in TERT-promoter-mutant glioma and other cancers. GABP binds DNA as a heterodimer or a heterotetramer which regulate functionally distinct transcriptional programs. In our preliminary data, we identify a specific heterotetramer forming GABP?1 isoform (GABP?1L) that is dispensable in normal cells but may be critical for mutant TERT promoter activation and tumor cell immortalization. If the mutant TERT promoter is uniformly present throughout each tumor, and if GABP?1L modulation leads to tumor cell death while sparing normal cells, the GABP pathway may represent a new therapeutic option for mutant TERT promoter-driven malignancies. We will test this hypothesis with three specific aims: In Aim 1, we will determine the extent to which the TERT promoter mutation is clonal at diagnosis and recurrence. We devised a robust system to collect and analyze clonality in 10 spatially mapped samples from each GBM and OD, representing maximal tumor geography. In Aim 2, we will determine if the GABP heterotetramer is required to maintain cellular immortality in TERT promoter mutant CNS tumors. We will use CRISPR-Cas9 genetic targeting of the GABPB1L isoform to determine the consequences on TERT expression, telomere length, cell viability and tumor formation. The transcriptome effects and death mechanism of GABP?1L deficient tumor cells will be determined to identify vulnerabilities to exploit with existing therapies. In Aim 3, we will identify therapies that will increase cell death in TERT promoter mutant tumors deficient in GABP?1L. In our preliminary data, failure of GBM cells to fully activate TERT expression by a GABP heterotetramer culminates in telomere dysfunction and DNA damage. We will perform a focused, exploratory screen of DNA damaging and DNA damage response-inhibiting agents on GABP?1L deficient cells to identify therapies that will increase cell death and decrease tumor formation. These studies could establish the GABP?1L isoform as a valuable therapeutic target specifically for TERT promoter mutant CNS tumors, and potentially many others. In parallel, we will advance drug discovery and development efforts towards small molecule inhibitors of different GABP subunits with industry partners Telo Therapeutics and GlaxoSmithKline.